Establishing 3D Printed Microfluidics for Molecular Biology Workflows

The Idea

The basic tools of a molecular biologist have changed little since the early 1990s; procedures tend to be performed manually and involve the use of large volumes meaning progress is slow and low throughput. These factors become problematic as research begins to move into the era of whole genome engineering . Over the last decade there have been significant advances in the field of microfluidics leading to the production of lab-on-chip devices , but the downside of these approaches is that they tend to rely on expensive, specialist equipment, meaning they are out of reach to most molecular biology laboratories. Several recent publications have begun to address this issue, utilizing advances in 3D printing to create low cost alternatives . By utilizing expertise in Cambridge and the NBI (Norwich Biosciences Institutes), the aim of this proposal is to design and test a 3D printed, modular microfluidic setup for molecular biology. All designs will be documented and open access, and, by instigating a standard for modular microfluidics, the outputs can serve as a basis for further innovation.

Who We Are

Dr. Steven Burgess (CAM)– Molecular Biology

Dr. Tom Meany (CAM) – Microfluidics/ 3D printing/ Optics

Dr. Richard Bowman (CAM) – Optics/ 3D printed microscope

Dr. Oleg Raitskin (TSL) – Molecular Biology

Dr. Neil Pearson (TGAC)- 3D printing

Implementation

Part I:

The first step will involve adapting existing designs for 3D printed microfluidics to make them compatible with each other ; this will also require defining a standard for interfacing between modular microfluidic components. In developing this standard we will seek to consult with the wider synthetic biology standards and microfluidics communities.

In the initial stage we will build five modules with wide applications in molecular biology, using published designs as a starting point:

A microfluidic pump for movement of liquids.

A water droplet generator for encapsulation.

An optical setup for droplet analysis.

A component for isolating DNA based on magnetic beads .

A component for thermal cycling.

Part II:

Once the 3D printed components have been created, we will test the operation of the modules. Experiment one will be to test the pump, droplet encapsulation and microscope in combination for encapsulation of aqueous solutions. This has the potential for future applications in single cell analyses. Experiment two will test the ability of the pump, thermal cycler and DNA isolation component for the construction of plasmid constructs, by current cloning techniques such as golden-gate assembly. We intend to run several iterations of the design-build-test cycle to improve the functioning of components.

Benefits and outcomes

1. A standard for 3D printed modular microfluidics.

2. Five designs for compatible 3D printed modules for molecular biology workflows.

3. Protocols for droplet encapsulation and DNA assembly on 3D-printed microfluidic devices

4. DocuBrick (http://docubricks.com/) documentation for all parts. By characterising the designs and making them open access, it will facilitate use by the wider community.

Budget

Component                                                                              Cost

Microfluidic Pump: Consumables (3D printing)                  ~£100 per iteration

Water Droplet Generator: Consumables (3D printing)      ~£100 per iteration

Optics: Consumables (3D printing)                                        ~£200 per iteration

DNA isolation: Consumables (3D printing)                          ~£100 per iteration

Thermal Cycler: Consumables (3D printing)                       ~£100 per iteration

Total Cost per iteration                                                             £600 per iteration

Subtotal: (Cost for 4 iterations of each device plus duplication of final design for simultaneous testing of function in two laboratories)                                                      £2400

DNA isolation: Electromagnet                                                2 @ £ 100 each

Thermal Cycler: Diode Laser                                                   2 @ £ 600 each

Molecular biology reagents e.g.  MagJET Plasmid DNA Kit (96 preps; K2791), TypeIIS enzyme (ThermoFIsher BsaI ER0291)                                                  £200

Misc. costs e.g. package and shipment of 3D printed devices between Norwich and Cambridge                                                                                                                        £100

Total:                                                                                       £4100

Establish a Procedure for Rapid Identification of Genetic Parts for Use in Algal Biotechnology

The Idea

We propose to run a pilot experiment to investigate the feasibility of using DNase-SEQ to identify of regulatory elements in Chlamydomonas reinhardtii; with the view to producing a genetic toolkit for this alga. DNase-SEQ is a powerful approach to identify transcription factor (TF) binding sites (He et al. 2014) which can then be utilised as genetic parts. To date there have been no reports of DNase-SEQ being applied to C. reinhardtii so the first stage of the project will be to establish the procedure.

As a test case we will focus on identifying regulatory elements that control the induction of the algal carbon concentrating mechanism (CCM). The reason for this is that the Griffiths lab is interested to understand the CCM, and a set of genetic parts that induce expression upon CCM induction could serve as useful tools for future analyses, such as high throughput screening of carbon concentrating components which could be engineered into higher plants for crop yield improvement.

Additionally, exploring DNase-SEQ data can be difficult for those without bioinformatics experience; we therefore aim to develop an open access, online tool to facilitate this process.

Who We Are

Cindy Chan (kxc22@cam.ac.uk)

PhD student in Physiological Ecology group, Department of Plant Sciences, University of Cambridge under the supervision of Professor Howard Griffiths.

Current research focus is the carbon concentration mechanism (CCM) of Chlamydomonas reinhardtii with a focus on the pyrenoid biogenesis.

Role: In charge of cell culture and harvesting. Nuclei extraction and DNAse I digestion.

Dr. Steven Burgess (sjb287@cam.ac.uk)

Post-doctoral research associate in Molecular Physiology group, Department of Plant Sciences, University of Cambridge

Current research areas include identification of regulatory elements using DNase-SEQ

Role: Provide technical assistance in performing DNase-SEQ and a pipeline for DNase-SEQ analysis.

Marielle Vigouroux (marielle.vigouroux@jic.ac.uk)

Support specialist in computational and systems biology in John Innes Centre, Norwich

Current research includes the development of codon optimization tools in Chlamydomonas reinhardtii and analysis of RNA-Seq data.

Role: Generation of an online tool for exploration of DNase-SEQ data.

Implementation

Step 1: (3 months)

Synchronous culture of wild type reinhardtii cells at ambient air condition – Cindy

Cell harvest at five time points: -2D, -1D, 1L, 3L, 6L (D: dark, L: light) – Cindy

Nuclei harvest – Cindy & Steven

DNase I digestion – Cindy & Steven

Library preparation – sequencing service (Dept. of Pathology, Cambridge)

Step 2: (2 months)

Sequencing – sequencing service (Dept. of Pathology, Cambridge)

DNase-SEQ pipeline refinement – Steven

Data analysis – Cindy

Step 3: (6 months)

Development of webpage – Marielle

Note: We have some preliminary data which can be used to establish the online tool so that the webpage development will be run in parallel to the experimental part of the project.

Benefits and outcomes

A set of candidate light responsive sequences for use as genetic parts in C. reinhardtii.

A protocol for performing DNase-SEQ analysis in C. reinhardtii.

An open-source pipeline for DNase-SEQ analysis.

A biologist-friendly website of the regulatory network of carbon concentrating mechanism of Chlamydomonas reinhardtii which would be open to public access.

Budget

NextSeq 500 Mid Output: £1450.00

Library Preparation: £2500.00

Webpage Development: £50.00

Total: £4000.00

Advancing the ability to image single RNA molecules at the cellular level

The Idea

Plant biology currently lags behind other fields in the study of cell-to-cell variation and subcellular localization of mRNA. During my PhD I helped to establish the first Single molecule Fluorescent In situ hybridization (smFISH) method for plants where each RNA molecule can be visualized as a single fluorescent dot in Arabidopsis thaliana root meristem tissue (Duncan et al., Plant methods, 2016 in press). This technique, in combination with an image analysis algorithm developed by colleagues in the Computational and Systems Biology department at JIC, was successfully used to quantify mRNA at the cellular level. It also revealed subcellular localisation of coding and non-coding RNA and provided data to enable the estimation of the frequency of transcriptional firing events. The high level of back ground autofluorescence emitted by many green plant tissues currently limits smFISH analysis to a single tissue type. With the support of OpenPlant we propose to promote and optimise this existing technique. In addition, we aim to adapt the methodology for use in other Arabidopsis tissues and to enable RNA imaging in the liverwort Marchantia polymorpha.

Who We Are

Dr Susan Duncan, Postdoctoral Researcher, Grieneisen Lab, Department of Computational and Systems Biology, John Innes Centre, Norwich.

Email: susan.duncan@jic.ac.uk.

I am a cell biologist researching Arabidopsis cell polarity. I have relevant smFISH expertise.

Dr Susana Sauret-Gueto, Research Associate and Lab Manager, Jim Haseloff group, Department of Plant Sciences, University of Cambridge.

Email: ss2359@cam.ac.uk.

I work on developing foundational technologies, protocols and workflows for the engineering of the model plant Marchantia polymorpha. I oversee instrumentation, including microscopes, in the OpenPlant Lab in Cambridge

Mr Christian R. Boehm, Doctoral Researcher, Department of Plant Sciences, University of Cambridge.

Email:crb59@cam.ac.uk.

I am a synthetic biologist working on the development of genetic circuits controlling transgene expression in Marchantia polymorpha. My expertise relevant to this proposal embraces maintenance, propagation and transformation of M. polymorpha, the development of circuits for transgene expression and quantitative confocal microscopy.

Implementation

We propose three specific aims:

1. Establish the existing smFISH technique in Cambridge and optimise imaging

Training scientists to use this technique in Cambridge will effectively promote its use across the plant science community. Also, observing similar A. thaliana samples using different microscopes, fluorescent filter sets and cameras available in Cambridge will test the robustness of the system and aid understanding of existing imaging limitations.

2. Development of two modified smFISH approaches

Two separate approaches will be used to detect amplified mVenus mRNA fluorescent signals in Arabidopsis root meristem cells. The first will use branched DNA probes together with a brighter dye (similar to Sinnamon and Czaplinski, Methods Mol. Biol 2015). The second will use multiple branched DNA probes together with a bright, stable dye to amplify the signals up to 100X (Battich et al, Cell2015).

3. Evalute the two smFISH approaches

The two smFISH approaches will be tested on Arabidopsis leaves and hypocotyls, in addition to Marchantia gemmae. Tissue clearing techniques can also be employed, if necessary, to observe RNA signals.

Outcome

This project aims to promote and advance the use of smFISH in studies of plant gene regulation. It will provide a clearer understanding of current imaging limitations together with the first co-ordinated attempt to expand the methodology into other plant tissues and species. The knowledge gained as part of this project will prove invaluable for ongoing work even if attempts to widen its applicability have limited success. It also has the potential to guide future plans to develop smFISH as a high throughput methodology.

Benefits and outcomes

Enhancing an existing smFISH protocol supports the OpenPlant remit as this open technology provides invaluable insights into plant gene regulation. As genetic engineering projects become more ambitious, such understanding will become increasing important and could play a key role in developing strategies to mitigate transgenic gene silencing issues. This project complements existing work in both laboratories. The smFISH technique is established in the Grieneisen lab at The John Innes Centre and both microscopy resources and Marchantia expertise are central to work carried out in the Haseloff laboratory in Cambridge.

We plan to publically document outcomes from this project:

a.    A YouTube video of the existing smFISH technique will be made freely available to promote this method more widely across the plant science community.

b.    The images generated by different microscopes in Norwich and Cambridge will be made freely available online to help other researchers decide on the suitability of their equipment before embarking on smFISH experiments.

c.    The project will lead to publication in a peer-reviewed journal if we achieve our aim of adapting smFISH for use in other Arabidopsis tissues and Marchantia gemmae.

Budget

Approach 1 Probes (IDT): £700

Approach 2 Probes (Affymetrix): £1000

Other consumable costs: £600

Microscopy expenses (JIC): £960

Travel to Cambridge / Norwich: £140

Total: £4000